Development The Indirect ELISA Based On The Prokaryotic Expression Of VP7Gene Of African Sickness Virus

This study successfully prokaryotic expresses a group-specific recombinant protein VP7ofAfrican horse sickness virus and develops an indirect ELISA for the detection of antibodies toVP7in all nine horse serotypes. This method is specific and sensitive.Compared with the standardforeign ELISA，the coincidence rate between the two methods was100%.The experiment aimed at the group-specific protein VP7. Comparing all amino acidssequences of AHSV-VP7in GenBank,choose the most conservative region was chosen and thecodes were optimized to increase the expression quantity. One pair of specific primers weredesigned and synthesized to obtain the AHSV-VP7gene by PCR. The AHSV-VP7gene was thencloned into the plasmid pMD18-T,and double digested by EcoR I and Hind III. The recombinantexpression plasmid pET-30a-VP7was constructed and transformed into Rosetta（DE3）. The resultsshowed that the protein of44ku was highly expressed by the optimized conditions. The westernblot and SDS-PAGE analysis confirmed that the protein was highly expressed and had goodantigenicity. Purification of the target protein by His·Bind kits and confirms that the purifiedproteins have antigen activity.The purified recombinant VP7protein as detection antigen was usedto establish a detection method to determine serum antibody against AHS virus, and the reactionconditions in the indirect ELISA such as antigen coating concentration, the dilution of sera andreaction time were optimized. The optimal test showed that the antigen coating concentration was0.625ng/μL, the dilution of serum was1:50, the optimal time was30min, optimal dilution ofanti-HRP was1:40000,the optimal time was45min and the cut off values was0.36respectively.Compared with the standard competitive ELISA，the coincidence rate between the two methodswas100%.The established indirect ELISA was then used to detect serum antibody against AHSvirus, which indicated that the method was sensitive and specific.There are not kits on AHSV detection in china at present and to establish a rapid specificdiagnostic method of our own is imperative.The study establishes a simple and convenientserological detection method for AHSV and settles a foundation for the development of diagnosticELISA assay kits to distinguish antibodies of AHSV.